Phenolic Profile of Herbal Infusion and Polyphenol-Rich Extract from Leaves of the Medicinal Plant Antirhea borbonica: Toxicity Assay Determination in Zebrafish Embryos and Larvae

Abstract

Antirhea borbonica (A. borbonica) is an endemic plant from the Mascarene archipelago in the Indian Ocean commonly used in traditional medicine for its health benefits. This study aims (1) at exploring polyphenols profiles from two types of extracts-aqueous (herbal infusion) and acetonic (polyphenol rich) extracts from A. borbonica leaves-and (2) at evaluating their potential toxicity in vivo for the first time. We first demonstrated that, whatever type of extraction is used, both extracts displayed significant antioxidant properties and acid phenolic and flavonoid contents. By using selective liquid chromatography-tandem mass spectrometry, we performed polyphenol identification and quantification. Among the 19 identified polyphenols, we reported that the main ones were caffeic acid derivatives and quercetin-3-O-rutinoside. Then, we performed a Fish Embryo Acute Toxicity test to assess the toxicity of both extracts following the Organisation for Economic Cooperation and Development (OECD) guidelines. In both zebrafish embryos and larvae, the polyphenols-rich extract obtained by acetonic extraction followed by evaporation and resuspension in water exhibits a higher toxic effect with a median lethal concentration (LC₅₀: 5.6 g/L) compared to the aqueous extract (LC₅₀: 20.3 g/L). Our data also reveal that at non-lethal concentrations of 2.3 and 7.2 g/L for the polyphenol-rich extract and herbal infusion, respectively, morphological malformations such as spinal curvature, pericardial edema, and developmental delay may occur. In conclusion, our study strongly suggests that the evaluation of the toxicity of medicinal plants should be systematically carried out and considered when studying therapeutic effects on living organisms.

Keywords: Antirhea borbonica; LC-MS/MS; medicinal plants; polyphenols; toxicity; zebrafish.

Figure 1

Antioxidant capacity, total phenolic acid, and flavonoid contents from A. borbonica extracts. (A) Total antioxidant capacity of polyphenols-rich extracts from A. borbonica was measured by a the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. Ascorbic acid was used as a positive control. The results are expressed as the % of reduced DPPH. (B) Total phenolic contents of acetonic and aqueous extracts from A. borbonica were determined using the Folin–Ciocalteu colorimetric assay at a concentration of 40 g/L (plant dried powder). The results are expressed as the mg gallic acid equivalent (GAE)/100 g of plant dried powder. (C) Total flavonoid contents were determined using an aluminum chloride colorimetric assay. The results are expressed as the mg quercetin equivalent (QE)/100 g of plant dried powder. Data are the means ± SDs of three independent experiments. * p < 0.05, *** p < 0.001 (vs. 40 g/L of acetonic extract), and ^$$ p < 0.01 (vs. 40 g/L (GAE) ascorbic acid).

Figure 2

Spectra obtained for a representative A. borbonica acetone-evaporated extract. (A) Representative total ion chromatogram (TIC) obtained in negative mode. (B) UHPLC-UV chromatograms obtained at 280 and 310 nm (C). The different molecules are numbered according to their retention times.

Figure 3

Survival curves for 96 hpf zebrafish embryos exposed with acetonic (A) or aqueous (B) extracts from A. borbonica at high concentrations of 16.9 g/L (acetonic) and 40 g/L (aqueous) and a low concentration of 2.3 g/L (acetonic and aqueous), and E3 was considered as control. Median lethal concentration curves (LC₅₀) for 96 hpf zebrafish embryos and 72 hpf larvae exposed to acetonic (C,E) or aqueous (D,F) extracts at different concentrations ranging from 16.9 to 1.3 g/L (acetonic) and 40 to 2.3 g/L (aqueous) for 4 and 2 days, respectively. The LC₅₀ values were expressed in g/L. Data are the mean ± SD of three independent experiments.

Figure 4

Morphological malformations and delayed development of zebrafish embryos/larvae exposed to A. borbonica extracts. (A) From left to right, coagulated egg (at 24 hpf), delayed hatching, spinal curvature, pericardial edema, and control embryos/larvae (96 hpf). Arrows indicate the presence of pericardial edema (PE) and spinal curvature (SC). Hatchability rates after 4 days of exposure with acetonic and aqueous extracts at 2.3, 7.2, and 9.5 g/L are represented in (B–D) represent the percentage of PE and SC, respectively. E3 medium was used as a positive control. Data are the mean ± SD of three independent experiments. ^$$$ p < 0.001 (vs. E3 (acetonic)), *** p < 0.001 (vs. acetonic extract), and ^### p < 0.001 (vs. E3 (aqueous)).